Summary MitoNEET, a primary target of the type II diabetes drug pioglitazone, is a key regulator of energy metabolism in cells. Structurally, mitoNEET is a homodimer with each monomer hosting a [2Fe-2S] cluster via three cysteine and one histidine residues in the C-terminal cytosolic domain. The N-terminus of mitoNEET contains a transmembrane ?-helix which anchors the protein to the mitochondrial outer membrane. Misregulation of mitoNEET has been implicated in causing type II diabetes, neurodegenerative diseases, and various types of cancers. However, the molecular mechanism by which mitoNEET regulates energy metabolism in cells remains largely elusive. Preliminary studies have shown that mitoNEET has a specific interaction with the reduced flavin mononucleotide (FMNH2) and that FMNH2 can quickly reduce the mitoNEET [2Fe-2S] clusters under aerobic or anaerobic conditions. On the other hand, the reduced mitoNEET [2Fe-2S] clusters can be reversibly oxidized by oxygen or ubiquinone. Furthermore, the type II diabetes drug pioglitazone analog NL-1 can effectively inhibit the electron transfer activity of mitoNEET. The goal of this application is to test a hypothesis that mitoNEET is a novel redox enzyme catalyzing electron transfer from FMNH2 to oxygen or ubiquinone in mitochondria and further to evaluate the physiological role of mitoNEET in cultured breast cancer cells. Aim 1 is to elucidate the molecular mechanism for the electron transfer in mitoNEET. The proposed research aims to determine the specific binding sites of FMNH2 and ubiquinone in mitoNEET by combining the AutoDock screening and site- directed mutagenesis with the electron paramagnetic resonance (EPR) spectroscopy and the electron transfer analyses. Possible reactive oxygen species and semiquinone intermediate formed during the electron transfer of mitoNEET under aerobic conditions will also be investigated using the rapid freeze-quench EPR spectroscopy. Aim 2 is to explore the physiological role of the electron transfer activity of mitoNEET in breast cancer cells. Previous studies have shown that mitoNEET is highly expressed in breast cancer cells, and depletion of mitoNEET significantly inhibits cancer cell proliferation. As FMNH2 is reduced by flavin reductase using NADH as the electron donors, mitoNEET may enhance glycolysis by promoting oxidation of NADH in cytosol. The proposed research in this aim is to explore the electron transfer activity of mitoNEET in mitochondria isolated from cultured breast cancer cells and to evaluate the glycolysis activity in the cells with or without depletion of mitoNEET. Success of the proposed research is expected to reveal the novel electron transfer activity of mitoNEET in mitochondria, to illustrate the physiological role of the electron transfer activity of mitoNEET in breast cancer cells, and to help develop therapeutic approaches for the mitoNEET-related human diseases. The proposed research will also provide opportunities for undergraduate and graduate students to have hands-on research experience and to make meaningful contributions to biomedical research projects.